Cell polarity is essential for development and differentiation, and it plays vital roles in fundamental processes such as cell migration, nutrient transport across epithelia, and neuronal transmission. Defects in cell polarity are often associated with serious human diseases such as cancer. Our long-term objective is to use the genetically tractable eukaryote Saccharomyces cerevisiae to elucidate the principles of cell polarization. In this proposal, we will address two important questions regarding the central role of Cdc42p, an evolutionarily conserved small GTPase, in polarity development. In Aim1, we will determine how Cdc42p activity is spatiotemporally regulated by its GAPs, Rga1p and Bem2p, in the formation of a single polarization domain, a common issue among all polarization systems. There are four GAPs (Rga1p, Rga2, Bem3p, and Bem2p) known to act on Cdc42p. Previously, we and others have shown that three GAPs (Rga1p, Rga2, and Bem3p) share a role in septin ring assembly at the beginning of the cell cycle. Recently, we discovered that Rga1p is uniquely required for preventing polarization within old cell division sites in post-cytokinesis cells. In this proposal, we will test our novel idea that Rga1p and Bem2p function redundantly at the bud neck as gatekeepers to restrict active Cdc42p and Rho1p to a single polarization domain, the bud cortex. To date, our studies have indicated that different GAPs can act alone or together to regulate specific cellular processes involving Cdc42p, and have helped establish a paradigm for studying complex regulation of mammalian Rho GTPases (~17 in human genome) by their numerous GAPs (~68 Rho GAPs in human genome). In Aim2, we will propose experiments to test our integrative model for the role of Cdc42p in polarized actin and septin organization, which together determine the overall cell shape and dictate their diverse functions in fundamental processes such as polarized exocytosis and directed cell migration. Our model states that Cdc42p controls polarized actin and septin organization via two genetically separable, but biochemically cross-talking pathways, one involving the evolutionarily conserved polarisome that is centered on the formin Bni1p, scaffold protein Spa2p, and the Rab GAPs Msb3p and Msb4p, and the other involving the yeast-specific Cdc42p effectors Gic1p and Gic2p. In this proposal, we will determine how the polarisome pathway affects septin organization via the actin cytoskeleton, how the Gic pathway affects actin organization via the septins, and how these two pathways crosstalk under cellular stresses such as 370C via Spa2p-based interactions. Homologues of Cdc42p are involved in numerous cellular functions such as cell polarity, cell migration, and cell growth control. Deregulation of Cdc42p activity in mammals is associated with serious human diseases, such as cancer. Thus, studying the signaling mechanisms of Cdc42p in yeast will have profound implication in basic biology and clinical sciences. PUBLIC HEALTH RELEVANCE: Cdc42p, an evolutionarily conserved small GTPase, plays essential roles in diverse cellular processes such as cell polarity, cell migration, and cell growth control. Deregulation of Cdc42p activity in mammals is associated with serious human diseases, such as cancer. Thus, studying the signaling mechanisms of Cdc42p in yeast will have profound implication in basic biology and clinical sciences.